Extracts from sophora species, method for producing the same and their use

ABSTRACT

The invention relates to extracts from Sophora species, to a method for producing the same and to their use in the prophylaxis and therapy of pathological conditions that are caused by estrogen deficiency or by other hormonal dysregulations.

[0001] The present invention relates to extracts of Sophora species,method for producing the same and their use for the prophylaxis andtreatment of pathological conditions caused by oestrogen deficiency orby dysregulations to sex-hormone-related metabolism, in particularoestrogen metabolism.

[0002] Irregular menstrual cycles appear in women from around the age of40, marking the onset of the menopause. This phase of changes in theendocrine system, known as the climacteric, may persist for a decade ormore. It results from the exhaustion of follicle growth and reducedresponses to gonadotrophin. As a consequence of this follicle deficit,production of oestrogen declines and eventually stops altogether. Inassociation with this, a variety of symptoms may develop, such as hotflushes, depression, anxiety, mental confusion, insomnia etc. Inaddition to these, serious health problems may arise as a result of thedecline in oestrogen production, such as osteoporosis, cardiacinsufficiency, cerebral infarction (strokes) and cancer.

[0003] Although hormone replacement therapy with oestrogens for therelief of climacteric-related complaints is highly effective, numerousstudies show that an increased risk of breast cancer and cancer of thewomb, cardiovascular diseases and changes to liver metabolism can belinked to this. The risk of cancer can be reduced by administeringprogestines. However, the protective effect of progestines appears tofade with long-term use and it is thought that hormone replacementtherapy (HRT) is linked with a 35-40% increased risk of breast cancer.Given the side effects, also distinct doubts as to the reliability ofthis type of treatment, the use of HRT medicine in post-menopausal womenover age 40 tends to be refused. It is estimated that only around 20% ofpost-menopausal American women receive HRT, of whom just 40% continuewith the treatment for longer than one year.

[0004] Substances that have an oestrogen-agonistic effect in humans oranimals, or which interact with oestrogen, have been discovered in manyplants. At least 20 different groups of substances withoestrogenic/anti-oestrogenic properties, coming from over 300 plants ofmore than 16 families of plants, have thus far been found. The majorityof the known phyto-oestrogens belong to the isoflavone, lignane orcumestane families. Only recently, a further highly potentphyto-oestrogen has been identified—8-prenylnaringenine (S. R. Milliganet al; Journal of Clin. Endocrinol. Metab. 84, 2249-2252 [1999]). Theoestrogenic potency of 8-prenylnaringenine in vitro was tested bystimulating alkaline phosphatase in Ishikawa-Var-I cells. In so doing,it was found that 8-penylnaringenine was significantly more active thanthe long-familiar phyto-oestrogens such as cumesterol, genistone ordaidzin, and produced only a slightly weaker effect than 17β oestradiol.

[0005] Controlled epidemiological studies on populations in the USA,Europe, China and Japan have shown that a close negative correlationexists between the uptake of phyto-oestrogens with food and theappearance of various tumours, especially breast cancer and prostatecancer. The inhibiting effects of phyto-oestrogens on the growth ofmammary and prostate tumour cells have also been confirmed in animalexperiments. The preventive properties of phyto-oestrogens are clearlybased on a large number of different biological characteristics thatdiscriminate between these combinations of synthetic and naturaloestrogens. Depending on the dosage and the endogenous hormone status,phyto-oestrogens also have oestrogenic or even anti-oestrogenic effects.Other biological effects include, for example, the inhibition oftyrosinkinases, DNA topoisomerases, ribosomal S6 kinase,ornithin-decaroxylase, aromatase or 5α reductase. Moreover,phyto-oestrogens frequently have anti-oxidising properties and it isclear that the sum total of all these various actions plays a part ininhibiting the appearance and growth of tumours (Tham et al, Journal ofClin. Endocrin. Metab. 83, 2223-2235 [1998]).

[0006] The object underlying the present invention is to provide plantextracts that are suitable for the prophylaxis and treatment ofpathological conditions that are caused by an oestrogen deficiency or bya dysregulation in norma sex-related hormone metabolism, in particularoestrogen metabolism.

[0007] A further purpose of the present invention is to provide a methodfor producing the extracts.

[0008] These objects are solved according to the invention by extractsfrom the Sophora species according to claims 1-9, the method forproducing the same according to claims 10-20, the pharmaceuticalpreparation according to claim 21, together with the use of the extractsaccording to claims 22-24.

[0009] Several Sophora species (Fabacaea) are known from traditionalChinese medicine whose roots can be used medicinally. These are Sophoraflavescens, S. subprostrata, S. alopeculoides, S. japonica, S.tonkinensis, S. tomentosa, S. moorcroftiana and S. leachiana. Of these,S. flavescens turns out to be of special significance. For this reason,it is found in the Chinese Pharmacopoeia (Sophorae flavescentis radix,“Kushen”, Pharmacopoeia of Chinese Medicine 9/1997) and is traditionallyused for the treatment of diarrhoea, gastro-intestinal haemorrhaging andeczema (W. Tang, G. Eisenbrand, Chinese Drugs of Plant Origin, SpringerVerlag, Berlin 1992).

[0010]S. flavescens roots, as well as those of other Sophora species,contain a sequence of chinolizidine alkaloids as one of their mainingredients. The Chinese Pharmacopoeia stipulates that the totaltitrimetric content of alkaloids may not fall below 2%. The mainalkaloids are matrine and oxymatrine.

[0011] Besides the alkaloids, a large number of flavones and relatedcompounds and triterpensaponines were found in the roots of S.flavescens, as well as in quite a few other Sophora species.

[0012] A variety of pharmacological properties have been described forSophora flavescens, also for individual ingredients or groups ofingredients. Alkaloids, in particular, have been thought to account foranti-arrhythmic, anti-histamine and anti-tussive effects, also foranti-ulcer effects (Tang and Eisenbrand, see above). Anti-neoplasticeffects and immune-suppressant properties have also been observed.

[0013] M. Kuroyanagi et al, Journal of Natural Products 1999, 62 (12)1595-1599, report on anti-bacterial and anti-androgenic flavonoids fromSophora flavescens. It was found that virtually all prenylflavonederivatives isolated from Sophora flavescens showed an anti-androgeniceffect.

[0014] W. M. Mazur et al, Nutritional Biochemistry 1998, 9, 193-200,report on the quantitative determination of the isoflavonesformononetin, biochanine A, daidzein, genistone and cumestrol and thelignanes secoisolariciresinol and matairesinol in leguminosae seeds,when seeds of Sophora japonica were investigated among others.

[0015] In toxicology, S. flavescens and S. japonica are classified astoxic. In particular, alkaloids of the matrine type are thought to bepoisonous (T. Blaszczyk, Deutsche Apotheke Zeitung 2001, 141 [14],1687-1696). In clinical samples, toxic effects such as severepalpitations, dyspnoea and spasms were observed in the case of Sophoraalkaloids. In animal studies, toxic effects could be shown foroxymatrine (K.-C. Huang, The Pharmacology of Chinese Herbs, CRC press,Boca Raton, 1993).

[0016] EP 0 998 924 A1 describes an anti-tumour treatment which containscompounds obtainable from plants with a topoisomerase II-inhibitingaction. Thus, in example 8, a description of obtaining leachinol A fromSophora is included. In this example, a dry powder of Sophora wassubjected to sequential extraction with acetone and methanol underreflux. The acetone extract was concentrated at reduced pressure andmixed with water. The resulting mixture is subjected to incrementaldistribution extraction by the use of benzol, ethylacetate andn-butanol. The ethylacetate extract is concentrated at reduced pressureand, in this way, a residue is obtained. The residue thus obtained iscleansed by silica-gel chromatography, using a benzol-acetone solventmixture; in so doing, leachianol A is obtained. The ethylacetate extractdescribed in example 8 differs from the extract of this invention inthat extracts obtained according to the invention contain macchian, andmacchian glucoside is greatly enriched (approx. five-fold). In boththese compounds, we are dealing with ingredients that are relevant tothe effectiveness of the extract according to the invention, as theinventors were able to show for the first time with the aid of anoestrogen receptor-bonding assay according to example 5. EP 0 998 924 A1gives no suggestion of the use of extracts of Sophora species for theprophylaxis and treatment of pathological conditions that are caused byoestrogen deficiency or by dysregulation in sex-related-hormonemetabolism, in particular oestrogen metabolism.

[0017] species of Sophora particularly favoured for the presentinvention are Sophora flavescens and Sophora subprostrata, for theiraqueous-alcoholic root extracts were found to have surprisingly powerfuloestrogenic effects in the context of this invention. It was possible toenrich these aqueous-alcoholic extracts by further separation andpurification. It turned out that pharmacological effects observed werecaused by the interaction of the isoflavones present in the extract:genistone and daidzein, flavones such as 8-prenylnaringenine, kushenolX, 8-prenylcamphorol, leachianon G and kushenol E, chalcones andpterocarpanes such as macchian and macchian glucoside, as well as otherunidentified flavonide-like compounds. In this connection, the chalconenewly discovered by the inventors: 2, 4, 4′, 6′tetrahydroxy-3′-lavandulyl-2′-methoxychalcone of the formula belowshould also be mentioned.

[0018]FIG. 1 illustrates the oestrogenic action of 70% [v/v] (62% [w/w])ethanol extracts (according to examples 1a) and 2a)) from Sophoraflavescens and Sophora subprostrata in a yeast assay, compared with 17βoestradiol.

[0019]FIG. 2 illustrates the oestrogenic action of extracts according tothe invention (70% [v/v] or 62% [w/w] ethanol extracts afterdistribution with ethylacetate; according to examples 1b) and 2b)) fromSophora flavescens and Sophora subprostrata in a yeast assay, comparedwith 17β oestradiol.

[0020]FIG. 3 illustrates the oestrogenic action of extracts according tothe invention (60% [w/w] ethanol extracts after distribution withethylacetate according to example 3) from Sophora flavescens in a yeastassay, compared with 17β oestradiol.

[0021] Similar extracts can generally be obtained by extraction with asolvent of medium polarity, chosen from the group consisting of aqueousalcohols, aqueous ketones and esters, including aqueous orwater-saturated esters, draining off the solvent and subsequentfluid-fluid distribution between an organic solvent and water.

[0022] According to the invention, an extract of Sophora species isprovided, obtainable by:

[0023] a) one or more extractions from Sophora species, using a solventselected from the group consisting of:

[0024] i) aqueous alcohols and aqueous ketones; and

[0025] ii) esters, including aqueous or water-saturated esters;

[0026] b) draining off the solvent from the alcoholic or ketonicsolution obtained at step a)i);

[0027] c) i) one or more distributions of the residue obtained at stepb) between an organic solvent selected from the group consisting ofethylacetate, tert-butylmethylether, n-butanol and butanon and water,and combining the solvent phases thus obtained; or

[0028] c) ii) one or more direct distributions of the ethylacetateextract obtained at step a)ii) with water; and

[0029] d) concentrating the combined organic solvent-phases obtained inc) till dry.

[0030] The ratio of drug to solvent at each extraction is in the rangeof around 1:7 to 1:10.

[0031] In a preferred embodiment, extraction at step a) is performedtwice.

[0032] The solvent used at step a) should preferably be selected fromthe group consisting of aqueous ethanol, aqueous acetone and aqueous orwater-saturated ethylacetate. In particular, the solvent used at step a)is 70% [v/v] (62% [w/w]) ethanol, 60% [w/w] ethanol or water-saturatedethylacetate.

[0033] The alcohols or ketones used at step a)i) are preferably appliedas 10-96% [v/v] or [w/w/], or 10-99% [v/v] or [w/w], particularly 50-92%[v/v] or [w/w] aqueous mixtures.

[0034] At step c)i), ethylacetate is the preferred solvent.

[0035] At step c) of the procedure, what takes place is essentially athinning-out of the total alkaloid content. The extract obtainable underthe invention is characterised in that it has a total alkaloid contentbelow 0.2%, and preferably below 0.1%. In particular, the extractobtained under the invention is free of alkaloid. In this context, theterm “alkaloid-free” means that it is undetectable using standardanalytical procedures such as HPLC. Furthermore, the extract under theinvention is characterised in containing flavones, isoflavones,chalcones and pterocarpanes. Flavone compounds can be isoprenylated,such as prenyl derivatives of naringenine. Isoflavone compounds may, ifnecessary, be mono-, bi- or tri-hydroxy substituted or O-methylated. Inaddition, the extract may contain glycosidated flavone and isoflavonecompounds. In particular, the extract according to the inventioncontains macchian, macchian glucoside, 8-prenylnaringenine, kushenol X,8-prenylcamphorol, leachianon G, kushenol E and 2,4,4′,6′ tetra-hydroxy3′ lavandulyl 2′ methoxychalcone.

[0036] The extract can be produced from one of the family of Sophoraspecies comprising Sophora flavescens, Sophora subprostrata, Sophoraalopeculoides, Sophora japonica, Sophora tonkinensis, Sophora tomentosa,Sophora moorcroftiana and Sophora leachiana; preferably Sophoraflavescens or Sophora subprostrata.

[0037] The extracts obtainable according to the invention can beprocessed together with conventional pharmaceutically-acceptableadditives to pharmaceutical preparations such as capsules, film tabletsor coated tablets, wherein as conventional pharmaceutically-acceptableadditives fillings, bonding agents, spreaders and coatings for film andcoated tablets, as well as oil or fat as excipients for capsules, can beused.

[0038] The extracts obtainable under the invention can be used for theprophylaxis and treatment of pathological conditions caused by oestrogendeficiency or by dysregulations to sex-hormone-related metabolism, inparticular oestrogen metabolism.

[0039] The pathological conditions can be selected from those belongingto the group consisting of climacteric complaints, sex hormone-dependentcancers, benign prostate hyperplasia, osteoporosis, Alzheimer's diseaseand cardiovascular diseases: the cancers included here are breastcancer, prostate cancer and cancer of the womb.

[0040] The extracts according to the invention are applied to humans ata dosage from 1-1000 mg daily, preferably 100-1000 mg daily.

[0041] A treatment is made available by using the combination accordingto the invention that includes flavone, isoflavone, chalcone,pterocarpane and other similar compounds that is particularly suitablefor combating pathological conditions caused by oestrogen deficiency orby dysregulations to sex hormone-related metabolism, in particularoestrogen metabolism.

[0042] The following examples describe the invention and should not beconsidered to limit the invention. All percentage details refer to theweight, unless specified otherwise.

EXAMPLE 1 Production of an Extract From Sophora flavescens

[0043] a) Extraction

[0044] 2 kg of ground Sophora flavescens roots were mixed with 14 kg 70%[v/v] (62% [w/w]) ethanol, extracted by spinning (Ultraturrax) for 5 minand then stirred intensively for 1 hr at 50° C. Then it was filteredwith suction over a Supra Seitz 1500 filter and the drug residue wasthen extracted for a second time in the same way. Both the extractsolutions were combined and the dry extract content was determined usingan aliquot. It turned out that there was a dry residue of 475.6 g,corresponding to a yield of 23.8%.

[0045] b) Distribution

[0046] The ethanol was removed from the extract solution on the rotaryevaporator at 50° C. The watery residue (5 l) was stirred three timeswith 2 l ethylacetate each time, the ethylacetate phases were combinedand concentrated in the rotary evaporator till dry.$\underset{\_}{{Ethylacetate}\quad {extract}} = {67.84\quad g\quad {\left( {{3.4\% \quad {by}\quad {reference}\quad {to}\quad {the}\quad {drug}};\quad {14.3\% \quad {by}\quad {reference}\quad {to}\quad {total}\quad {extract}}} \right).}}$

[0047] According to the HPLC analysis, the ethylacetate extract containsflavones, isoflavones, chalcones, pterocarpanes (e.g.8-prenylnaringenine, daidzein, kushenol X, norkurarinon, macchian,genistone, 8-prenylcamphorol). There was no evidence of alkaloids.

[0048] This combination of substances is preferably suited to theprophylaxis of and treatment of pathological conditions caused byoestrogen deficiency or by dysregulations to sex hormone-relatedmetabolism, in particular oestrogen metabolism (compare example 5).

EXAMPLE 2 Production of an Extract From Sophora subprostrata

[0049] a) Extraction

[0050] 1 kg of ground S. subprostrata roots was mixed with 7 kg 70%[v/v] (62% [w/w]) ethanol, extracted by spinning (Ultraturrax) for 5minutes and then stirred intensively at 50° C. for 1 hour. Then it wasfiltered with suction over a Supra Seitz 1500 filter and the drugresidue was then extracted for a second time in the same way. Both theextract solutions were combined and the dry extract content wasdetermined using an aliquot. It turned out that there was a dry residueof 111.8 g, corresponding to a yield of 11.2%.

[0051] b) Distribution

[0052] The ethanol was removed from the extract solution on the rotaryevaporator at 50° C. The watery residue (2.5 l) was stirred thrice withle ethylacetate each time, the ethylacetate phases were combined andconcentrated in the rotary evaporator till dry.$\underset{\_}{{Ethylacetate}\quad {extract}} = {10.4\quad g\quad \left( {{1.0\% \quad {by}\quad {reference}\quad {to}\quad {the}\quad {drug}};\quad {9.3\% \quad {by}\quad {reference}\quad {to}\quad {total}\quad {extract}}} \right)}$

EXAMPLE 3

[0053] 36 kg of ground Sophora flavescens drug was mixed with 7.7 timesits weight made up of 60% (w/w) ethanol. Given vigorous stirring andwith Dispax (Ultraturrax) in circulation, the extract solution wasextracted for 1 hr at 50° C. The extract solution was filtered clear,the drug residue was once again extracted with 7 times its weight madeup of 60% (w/w) ethanol under the same conditions, and finally filtered.

[0054] The extract solution was then concentrated in the centrifugalevaporator to an ethanol content of 3.5% and dry extract content of10.52%. This concentrated solution was then distributed againstwater-saturated ethylacetate three times at a 1:0.4 ratio by volume. Theethylacetate phase was concentrated at the rotary evaporator and driedout in the drying cupboard at 60° C.

[0055] This resulted in:

[0056] Ethylacetate extract: 1.188 kg after grinding (3.3% by referenceto the drug).

[0057] HPLC content of pharmacologically relevant substances: Macchianglucoside 2.8% 8-prenylnaringenine 0.4% Macchian 0.6% Kushenol X 1.3%8-prenylcamphorol 0.7% Leachianon G 6.6% Kushenol E 0.3%2,4,4′,6′-tetrahydroxy-3′- 0.7% lavandulyl-2′-methoxychalcone

[0058] There was also evidence of daidzein and genistone. There was noevidence of any alkaloids.

[0059] Estrogenic action could be shown for each of the substancesmentioned in a yeast assay according to example 5. The oestrogenicaction for the extract is produced by the Reportergen assay with the useof yeast cells according to example 5: compare FIG. 3.

[0060] HPLC Method Columns LiChrosphere 100 5 μm, 250 × 4 mm Eluens A:1000 ml bidest water/3 ml phosphoric acid (85%)/2 ml triethylamine B:100 ml acetonitril/3 ml phosphoric acid (85%)/2 ml triethylamine/60 mlbidest water Gradient % A % B 0 min 70 30 30 min 30 70 35 min 0 100 Flow 1.2 ml/min Detection 220 mn

EXAMPLE 4

[0061] 150 g of ground Sophora flavescens drug was extracted twice withwater-saturated ethylacetate at the ratio of 1:7 (m/m). Therefore, thedrug was broken down beforehand with an Ultra-Turrax for five minutes(extraction by movement). The drug was then extracted under reflux for 1hour at 60° C. The combined extract solutions were then filtered off andthe ethylacetate extract solution thus obtained was shaken back twicewith ethylacetate-saturated water at the ratio 1:1 [v/v]. The combinedethylacatate phases were concentrated till dry.

[0062] Yield: 3.99% by reference to the drug.

EXAMPLE 5 Testing the Ethylacetate Extract Produced for OestrogenicAction With Transfected Yeast Cells, Which Express the Human a OestrogenReceptor

[0063] The testing of extracts for oestrogenic properties was performedwith a Reportergen assay, using yeast cells (saccharomyces). These cellsare stable with the human α oestrogen receptor and an expression plasmidwhich contains an oestrogen response element and the gene for the enzymeβ galactosidase. All samples were dissolved in DMSO at a concentrationof 20 mg/ml, and were given undiluted or after diluting with DMSO at theratio of 1/10, 1/100 or at a volume of 1 μl to 100 μl culture medium in96-well flat-bottom micro-titre dishes. Next, 100 μl yeast suspensionand the chromogenous substrate chlorophenolred-β-D-galactopyranosidewere added. Control wells were provided on every dish, which were filledwith either the culture medium or the solvent alone, or which containedthe standard concentrations of 17β oestradiol. The yeast cells wereincubated for 72 hours at 32° C., after which absorption of the mediumwas measured at 540 nM in a micro-titre dish photometer. The sampleswere sometimes checked twice.

[0064] Results:

[0065] Sample Concentrations: Dilution Concentration undiluted 100 μg/ml1:10   10 μg/ml 1:100  1 μg/ml

[0066] Designation of Samples: Sample Number of number Sample testsSophora species A 70% [v/v] (62% [w/w]) 1 Sophora flavescens ethanolextract according to ex. 1a) B 70% [v/v] (62% [w/w] 1 Sophorasubprostrata (ethanol extract according to ex. 2a) C 70% [v/v] (62%[w/w] 2 Sophora flavescens ethanol extract according to ex. 1a) DEthylacetate extract 1 Sophora flavescens according to ex. 1b) EEthylacetate extract 1 Sophora subprostrata according to ex. 2b) FEthylacetate extract 1 Sophora flavescens according to ex. 3

[0067] The results of the assays are depicted in FIGS. 1, 2 and 3. In sodoing, the oestrogenic action of the extracts was compared against theaction of 17β oestradiol. For this purpose, extracts that can bedesignated as “active” are those whose action when compared with 17βoestradiol matches the action of 17β oestradiol that has becomeestablished within the distinctly rising segment of the 17β oestradiolactivity curve (and hence from a concentration of around 0.1-0.2 nM of17β oestradiol).

[0068] In particular, FIG. 1 illustrates the oestrogenic action of 70%[v/v] (62% [w/w]) ethanol extracts (according to examples 1a) and 2a))from Sophora flavescens and Sophora subprostrata in a yeast assay,compared with 17β oestradiol.

[0069]FIG. 2 illustrates the oestrogenic action of the extracts underthis invention (70% [v/v] or 62% [w/w] ethanol extract afterdistribution with ethylacetate; according to examples 1b) and 2b)) fromSophora flavescens and Sophora subprostrata in a yeast assay, comparedwith 17β oestradiol.

[0070]FIG. 3 illustrates the oestrogenic action of extracts according tothe invention (60% [w/w] ethanol extracts after distribution withethylacetate according to example 3) from Sophora flavescens in a yeastassay, compared with 17β oestradiol.

1. Extract from Sophora species, characterised in that it contains notmore than 0.2% alkaloids in total, and that it contains flavones,isoflavones, chalcones and pterocarpanes, wherein the flavones include8-prenylnaringenine, kushenol X, 8-prenylcamphorol, laechianon G andkushenol E, wherein the isoflavones include daidzein and genistone,wherein the chalcones include2,4,4′,6′-tetrahydroxy-3′-lavandulyl-2′-methoxychalcone, wherein thepterocarpanes include macchian and macchian-glucoside.
 2. Extractaccording to claim 1, characterised in that it contains not more than0.1% alkaloids in total.
 3. Extract according to claim 1, characterisedin that it is completely free of alkaloids.
 4. Extract according to anyof claims 1 to 3, wherein the species of Sophora is selected from thegroup consisting of Sophora flavescens, Sophora subprostrata, Sophoraalopeculoides, Sophora japonica, Sophora tonkinensis, Sophora tomentosa,Sophora moorcroftiana and Sophora leachiana.
 5. Extract according to anyof claims 1 to 3, wherein the species of Sophora is selected fromSophora flavescens and Sophora subprostrata.
 6. Method for obtaining anextract from Sophora species, characterized by the steps: a) one or moreextractions from Sophora species with a solvent selected from the groupconsisting of i) aqueous alcohols and aqueous ketones; and ii) esters,including aqueous or water-saturated esters; b) draining off the solventfrom the alcoholic or ketonic extraction solution obtained at step a)i);c) i) one or more distributions of the residue obtained at step b)between an organic solvent selected from the group consisting ofethylacetate, tert-butylmethylether, n-butanol and butanon and water,and combining the solvent phases thus obtained; or c) ii) one or moredirect distributions of the extract obtained at step a)ii) with water;and d) concentrating the combined organic solvent phases obtained in c)till dry.
 7. Method according to claim 6, wherein extraction at step a)is performed twice.
 8. Method according to claim 6 or 7, wherein thesolvent used at step a)i) is aqueous ethanol.
 9. Method according toclaim 6 or 7, wherein the solvent used at step a)i) is aqueous acetone.10. Method according to any of claims 6 to 9, wherein the alcohol usedat step a)i) is present as 10-96% [v/v] or [w/w] or the ketone used atstep a)i) is present as 10-99% [v/v] or [w/w] aqueous mixture. 11.Method according to any of claims 6 to 9, wherein the alcohol used atstep a)i) or the ketone used at step a)i) is present as 50-92% [v/v] or[w/w] aqueous mixture.
 12. Method according to claim 6 or 7, wherein thesolvent used at step a)i) is 70% [v/v] or 60% [w/w] aqueous ethanol. 13.Method according to claim 6 or 7, wherein the solvent used at step a)ii)is ethylacetate or aqueous or water-saturated ethylacetate.
 14. Methodaccording to any of claims 6 to 13, wherein the solvent used at stepc)i) is ethylacetate.
 15. Method according to any of claims 6 to 14,wherein the species of Sophora is selected from the group consisting ofSophora flavescens, Sophora subprostrata, Sophora alopeculoides, Sophorajaponica, Sophora tonkinensis, Sophora tomentosa, Sophora moorcroftianaand Sophora leachiana.
 16. Method according to any of claims 6 to 14,wherein the species of Sophora is selected from Sophora flavescens andSophora subprostrata.
 17. Pharmaceutical preparation including anextract according to any of claims 1 to 5 and conventionalpharmaceutically-acceptable additives.
 18. Use of an extract accordingto any of claims 1 to 5, or a pharmaceutical preparation according toclaim 17, for the preparation of a medicament for the prophylaxis andtreatment of pathological conditions caused by a deficiency ofoestrogens or by a dysregulation of sex hormone metabolism, particularlyoestrogen metabolism.
 19. Use according to claim 18, wherein thepathological condition is selected from the group consisting ofclimacteric complaints, sex hormone-dependent cancers, benign prostatehyperplasia, osteoporosis, Alzheimer's disease and cardiovasculardiseases.
 20. Use according to claim 19, wherein the sexhormone-dependent cancers are selected from the group consisting ofbreast cancer, prostate cancer and cancer of the womb.
 24. Use accordingto claim 23, wherein the sex hormone-dependent cancers are selected fromthe group consisting of breast cancer, prostate cancer and cancer of thewomb.